Development of LC-MS/MS methods affording identification and measurement of active metabolites in rat and dog plasma after oral dosing of a penta-ethyl ester prodrug of diethylenetriamine pentaacetic acid

C2E5, the penta-ethyl ester prodrug of radionuclide decorporation agent diethylenetriamine pentaacetic acid (DTPA), was designed to address the logistical challenges of IV administration of DTPA in a mass casualty setting. The in vivo conversion of orally-dosed C2E5 to DTPA was evaluated in rat and dog plasma samples using LC-MS/MS methods developed with reference materials and stable-label internal standards for both analytes. C2E5 instability in plasma ex vivo was identified and addressed, but when C2E5 dosed samples revealed minimal C2E5 and DTPA, it became crucial to identify metabolites produced by degradation of C2E5 in vivo that could account for therapeutic efficacy reported. Development of an LC-MS/MS method that identified and estimated levels of eight de-esterified metabolites of C2E5 was initiated without availability of corresponding reference material by relying on predictions of their analyte-specific LC-MS/MS properties. Four de-esterified analogs of C2E5, suspected as active metabolites, were identified in rat or dog plasma. When complementary isomers, not in samples but in reference materials (including impurities identified), became available, they were used to estimate levels of the metabolites identified in dosed samples. Results affording measurement of C2E5 and metabolites in rat and dog plasma provided fit-for-purpose information that supported a timely advancement of the DTPA prodrug program.